Disruption and overexpression of 6-phosphofructo-2-kinase influence organic acid production in Aspergillus carbonarius ITEM 5010.

Aspergillus carbonarius is an efficient producer of organic acids with great potential for bio-based production of organic acids. In this study, we identified a gene f2kp encoding the enzyme 6-phosphofructo-2-kinase known as an allosteric regulator of the glycolytic flux and investigated its role in the production of organic acid. The strategy was to examine the impact of citric acid and malic acid production by overexpressing and disrupting f2kp, respectively. The overexpressing transformants expressed f2kp at higher level than the wild type, whereas no expression of f2kp was detected in the knockout transformants. Citric acid and malic acid production by the knockout strains decreased sharply along with a significant lower sugar consumption, though the overexpressing transformants produced similar amounts of citric acid and malic acid as the wild type. We conclude that 6-phosphofructo-2-kinase has an important regulatory role for the glycolytic flux and organic acid production in A. carbonarius.

Agrobacterium tumefaciens-mediated transformation of oleaginous yeast Lipomyces species.

Interest in using renewable sources of carbon, especially lignocellulosic biomass, for the production of hydrocarbon fuels and chemicals has fueled interest in exploring various organisms capable of producing hydrocarbon biofuels and chemicals or their precursors. The oleaginous (oil-producing) yeast Lipomyces starkeyi is the subject of active research regarding the production of triacylglycerides as hydrocarbon fuel precursors using a variety of carbohydrate and nutrient sources. The genome of L. starkeyi has been published, which opens the door to production strain improvements through the development and use of the tools of synthetic biology for this oleaginous species. The first step in establishment of synthetic biology tools for an organism is the development of effective and reliable transformation methods with suitable selectable marker genes and demonstration of the utility of the genetic elements needed for expression of introduced genes or deletion of endogenous genes. Chemical-based methods of transformation have been published but suffer from low efficiency. To address these problems, Agrobacterium-mediated transformation was investigated as an alternative method for L. starkeyi and other Lipomyces species. In this study, Agrobacterium-mediated transformation was demonstrated to be effective in the transformation of both L. starkeyi and other Lipomyces species. The deletion of the peroxisomal biogenesis factor 10 gene was also demonstrated in L. starkeyi. In addition to the bacterial antibiotic selection marker gene hygromycin B phosphotransferase, the bacterial ß-glucuronidase reporter gene under the control of L. starkeyi translation elongation factor 1α promoter was also stably expressed in six different Lipomyces species. The results from this study demonstrate that Agrobacterium-mediated transformation is a reliable and effective genetic tool for homologous recombination and expression of heterologous genes in L. starkeyi and other Lipomyces species.

Increased production of free fatty acids in Aspergillus oryzae by disruption of a predicted acyl-CoA synthetase gene.

Fatty acids are attractive molecules as source materials for the production of biodiesel fuel. Previously, we attained a 2.4-fold increase in fatty acid production by increasing the expression of fatty acid synthesis-related genes in Aspergillus oryzae. In this study, we achieved an additional increase in the production of fatty acids by disrupting a predicted acyl-CoA synthetase gene in A. oryzae. The A. oryzae genome is predicted to encode six acyl-CoA synthetase genes and disruption of AO090011000642, one of the six genes, resulted in a 9.2-fold higher accumulation (corresponding to an increased production of 0.23 mmol/g dry cell weight) of intracellular fatty acid in comparison to the wild-type strain. Furthermore, by introducing a niaD marker from Aspergillus nidulans to the disruptant, as well as changing the concentration of nitrogen in the culture medium from 10 to 350 mM, fatty acid productivity reached 0.54 mmol/g dry cell weight. Analysis of the relative composition of the major intracellular free fatty acids caused by disruption of AO090011000642 in comparison to the wild-type strain showed an increase in stearic acid (7 to 26 %), decrease in linoleic acid (50 to 27 %), and no significant changes in palmitic or oleic acid (each around 20-25 %).

Biosynthetic pathway for the epipolythiodioxopiperazine acetylaranotin in Aspergillus terreus revealed by genome-based deletion analysis.

Epipolythiodioxopiperazines (ETPs) are a class of fungal secondary metabolites derived from diketopiperazines. Acetylaranotin belongs to one structural subgroup of ETPs characterized by the presence of a seven-membered 4,5-dihydrooxepine ring. Defining the genes involved in acetylaranotin biosynthesis should provide a means to increase the production of these compounds and facilitate the engineering of second-generation molecules. The filamentous fungus Aspergillus terreus produces acetylaranotin and related natural products. Using targeted gene deletions, we have identified a cluster of nine genes (including one nonribosomal peptide synthetase gene, ataP) that is required for acetylaranotin biosynthesis. Chemical analysis of the wild-type and mutant strains enabled us to isolate 17 natural products from the acetylaranotin biosynthesis pathway. Nine of the compounds identified in this study are natural products that have not been reported previously. Our data have allowed us to propose a biosynthetic pathway for acetylaranotin and related natural products.

Increased production of fatty acids and triglycerides in Aspergillus oryzae by enhancing expressions of fatty acid synthesis-related genes.

Microbial production of fats and oils is being developed as a means of converting biomass to biofuels. Here we investigate enhancing expression of enzymes involved in the production of fatty acids and triglycerides as a means to increase production of these compounds in Aspergillus oryzae. Examination of the A. oryzae genome demonstrates that it contains two fatty acid synthases and several other genes that are predicted to be part of this biosynthetic pathway. We enhanced the expression of fatty acid synthesis-related genes by replacing their promoters with the promoter from the constitutively highly expressed gene tef1. We demonstrate that by simply increasing the expression of the fatty acid synthase genes we successfully increased the production of fatty acids and triglycerides by more than two-fold. Enhancement of expression of the fatty acid pathway genes ATP-citrate lyase and palmitoyl-ACP thioesterase increased productivity to a lesser extent. Increasing expression of acetyl-CoA carboxylase caused no detectable change in fatty acid levels. Increases in message level for each gene were monitored using quantitative real-time reverse transcription polymerase chain reaction. Our data demonstrate that a simple increase in the abundance of fatty acid synthase genes can increase the detectable amount of fatty acids.

New insight into the ochratoxin A biosynthetic pathway through deletion of a nonribosomal peptide synthetase gene in Aspergillus carbonarius.

Ochratoxin A (OTA), a mycotoxin produced by Aspergillus and Penicillium species, is composed of a dihydroisocoumarin ring linked to phenylalanine, and its biosynthetic pathway has not yet been completely elucidated. Most of the knowledge regarding the genetic and enzymatic aspects of OTA biosynthesis has been elucidated in Penicillium species. In Aspergillus species, only pks genes involved in the initial steps of the pathway have been partially characterized. In our study, the inactivation of a gene encoding a nonribosomal peptide synthetase (NRPS) in OTA-producing A. carbonarius ITEM 5010 has eliminated the ability of this fungus to produce OTA. This is the first report on the involvement of an nrps gene product in OTA biosynthetic pathway in an Aspergillus species. The absence of OTA and ochratoxin α, the isocoumaric derivative of OTA, and the concomitant increase of ochratoxin ß, the dechloro analog of ochratoxin α, were observed in the liquid culture of transformed strain. The data provide the first evidence that the enzymatic step adding phenylalanine to polyketide dihydroisocoumarin precedes the chlorination step to form OTA in A. carbonarius and that ochratoxin α is a product of hydrolysis of OTA, giving an interesting new insight into the biosynthetic pathway of the toxin.

Molecular genetic analysis reveals that a nonribosomal peptide synthetase-like (NRPS-like) gene in Aspergillus nidulans is responsible for microperfuranone biosynthesis.

Genome sequencing of Aspergillus species including Aspergillus nidulans has revealed that there are far more secondary metabolite biosynthetic gene clusters than secondary metabolites isolated from these organisms. This implies that these organisms can produce additional secondary metabolites, which have not yet been elucidated. The A. nidulans genome contains 12 nonribosomal peptide synthetase (NRPS), one hybrid polyketide synthase/NRPS, and 14 NRPS-like genes. The only NRPS-like gene in A. nidulans with a known product is tdiA, which is involved in terrequinone A biosynthesis. To attempt to identify the products of these NRPS-like genes, we replaced the native promoters of the NRPS-like genes with the inducible alcohol dehydrogenase (alcA) promoter. Our results demonstrated that induction of the single NRPS-like gene AN3396.4 led to the enhanced production of microperfuranone. Furthermore, heterologous expression of AN3396.4 in Aspergillus niger confirmed that only one NRPS-like gene, AN3396.4, is necessary for the production of microperfuranone.

Identifying and characterizing the most significant ß-glucosidase of the novel species Aspergillus saccharolyticus.

The newly discovered fungal species Aspergillus saccharolyticus was found to produce a culture broth rich in ß-glucosidase activity. In this present work, the main ß-glucosidase of A. saccharolyticus responsible for the efficient hydrolytic activity was identified, isolated, and characterized. Ion exchange chromatography was used to fractionate the culture broth, yielding fractions with high ß-glucosidase activity and only 1 visible band on an SDS-PAGE gel. Mass spectrometry analysis of this band gave peptide matches to ß-glucosidases from aspergilli. Through a polymerase chain reaction approach using degenerate primers and genome walking, a 2919 bp sequence encoding the 860 amino acid BGL1 polypeptide was determined. BGL1 of A. saccharolyticus has 91% and 82% identity with BGL1 from Aspergillus aculeatus and BGL1 from Aspergillus niger , respectively, both belonging to Glycoside Hydrolase family 3. Homology modeling studies suggested ß-glucosidase activity with preserved retaining mechanism and a wider catalytic pocket compared with other ß-glucosidases. The bgl1 gene was heterologously expressed in Trichoderma reesei QM6a, purified, and characterized by enzyme kinetics studies. The enzyme can hydrolyze cellobiose, p-nitrophenyl-ß-d-glucoside, and cellodextrins. The enzyme showed good thermostability, was stable at 50 °C, and at 60 °C it had a half-life of approximately 6 h.

Characterization of a polyketide synthase in Aspergillus niger whose product is a precursor for both dihydroxynaphthalene (DHN) melanin and naphtho-γ-pyrone.

The genome sequencing of the fungus Aspergillus niger uncovered a large cache of genes encoding enzymes thought to be involved in the production of secondary metabolites yet to be identified. Identification and structural characterization of many of these predicted secondary metabolites are hampered by their low concentration relative to the known A. niger metabolites such as the naphtho-γ-pyrone family of polyketides. We deleted a non-reducing PKS gene in A. niger strain ATCC 11414, a daughter strain of A. niger ATCC strain 1015 whose genome was sequenced by the DOE Joint Genome Institute. This PKS encoding gene we name albA is a predicted ortholog of alb1 from Aspergillus fumigatus which is responsible for production of the naphtho-γ-pyrone precursor for the 1,8-dihydroxynaphthalene (DHN) melanin/spore pigment. Our results show that the A. nigeralbA PKS is responsible for both the production of the spore pigment precursor and a family of naphtho-γ-pyrones commonly found in significant quantity in A. niger culture extracts. The generation of an A. niger strain devoid of naphtho-γ-pyrones will greatly facilitate the elucidation of cryptic biosynthetic pathways in this organism.

Alkane biosynthesis by Aspergillus carbonarius ITEM 5010 through heterologous expression of Synechococcus elongatus acyl-ACP/CoA reductase and aldehyde deformylating oxygenase genes.

In this study we describe the heterologous expression of the recently identified cyanobacterial pathway for long chain alkane biosynthesis, involving the reduction of fatty acyl-ACP to fatty aldehyde and the subsequent conversion of this into alkanes, in the filamentous fungus Aspergillus carbonarius ITEM 5010. Genes originating from Synechococcus elongatus strain PCC7942, encoding acyl-ACP/CoA reductase and aldehyde deformylating oxygenase enzymes, were successfully expressed in A. carbonarius, which lead to the production of pentadecane and heptadecane, alkanes that have not been previously produced by this fungus. Titers of 0.2, 0.5 and 2.7 mg/l pentadecane and 0.8, 1.6 and 10.2 mg/l heptadecane were achieved using glucose, Yeast malt and oatmeal media, respectively. Besides producing alkanes, we found elevated levels of internal free fatty acids and triglycerides in the alkane producing transformant. These findings can indicate that a yet unidentified, native fatty aldehyde dehydrogenase channels back the fatty aldehydes into the fatty acid metabolism, thus competing for substrate with the heterologously expressed fatty aldehyde deformylating oxygenase. These findings will potentially facilitate the future application of robust, fungal cell factories for the production of advanced biofuels from various substrates.

Spatial regulation of a common precursor from two distinct genes generates metabolite diversity.

In secondary metabolite biosynthesis, core synthetic genes such as polyketide synthase genes usually encode proteins that generate various backbone precursors. These precursors are modified by other tailoring enzymes to yield a large variety of different secondary metabolites. The number of core synthesis genes in a given species correlates, therefore, with the number of types of secondary metabolites the organism can produce. In our study, heterologous expression of all the A. terreus NRPS-like genes showed that two NRPS-like proteins, encoded by atmelA and apvA, release the same natural product, aspulvinone E. In hyphae this compound is converted to aspulvinones whereas in conidia it is converted to melanin. The genes are expressed in different tissues and this spatial control is probably regulated by their own specific promoters. Comparative genomics indicates that atmelA and apvA might share a same ancestral gene and the gene apvA is located in a highly conserved region in Aspergillus species that contains genes coding for life-essential proteins. Our data reveal the first case in secondary metabolite biosynthesis in which the tissue specific production of a single compound directs it into two separate pathways, producing distinct compounds with different functions. Our data also reveal that a single trans-prenyltransferase, AbpB, prenylates two substrates, aspulvinones and butyrolactones, revealing that genes outside of contiguous secondary metabolism gene clusters can modify more than one compound thereby expanding metabolite diversity. Our study raises the possibility of incorporation of spatial, cell-type specificity in expression of secondary metabolites of biological interest and provides new insight into designing and reconstituting their biosynthetic pathways.

Molecular genetic characterization of terreic acid pathway in Aspergillus terreus.

Terreic acid is a natural product derived from 6-methylsalicylic acid (6-MSA). A compact gene cluster for its biosynthesis was characterized. Isolation of the intermediates and shunt products from the mutant strains, combined with bioinformatic analyses, allowed for the proposition of a biosynthetic pathway for terreic acid.

Identification and characterization of the polyketide synthase involved in ochratoxin A biosynthesis in Aspergillus carbonarius.

Ochratoxin A (OTA) is a potent mycotoxin produced by Aspergillus and Penicillium species and is a common contaminant of a wide variety of food commodities, with Aspergillus carbonarius being the main producer of OTA contamination in grapes and wine. The molecular structure of OTA comprises a dihydroisocoumarin ring linked to phenylalanine and, as shown in different producing fungal species, a polyketide synthase (PKS) is a component of the OTA biosynthetic pathway. Similar to observations in other filamentous ascomycetes, the genome sequence of A. carbonarius contains a large number of genes predicted to encode PKSs. In this work a pks gene identified within the putative OTA cluster of A. carbonarius, designated as AcOTApks, was inactivated and the resulting mutant strain was unable to produce OTA, confirming the role of AcOTApks in this biosynthetic pathway. AcOTApks protein is characteristic of the highly reduced (HR)-PKS family, and also contains a putative methyltransferase domain likely responsible for the addition of the methyl group to the OTA polyketide structure. AcOTApks is different from the ACpks protein that we previously described in A. carbonarius, which showed an expression profile compatible with OTA production. We performed phylogenetic analyses of the ß-ketosynthase and acyl-transferase domains of the OTA PKSs that had been identified and characterized in different OTA producing fungal species. The phylogenetic results were similar for both domains analyzed and showed that OTA PKS of A. carbonarius, Aspergillus niger and Aspergillus ochraceus clustered in a monophyletic group with 100% bootstrap support suggesting a common origin, while the other OTA PKSs analyzed were phylogenetically distant. A quantitative RT-PCR assay monitored AcOTApks expression during fungal growth and concomitant production of OTA by A. carbonarius in synthetic grape medium. A clear correlation between the expression profile of AcOTApks and kinetics of OTA production was observed, with AcOTApks reaching its maximum level of transcription before OTA accumulation in mycelium reached its highest level, confirming the fact that gene transcription always precedes phenotypic production.

Engineering fungal nonreducing polyketide synthase by heterologous expression and domain swapping.

We reannotated the A. niger NR-PKS gene, e_gw1_19.204, and its downstream R domain gene, est_GWPlus_C_190476, as a single gene which we named dtbA. Heterologous expression of dtbA in A. nidulans demonstrated that DtbA protein produces two polyketides, 2,4-dihydroxy-3,5,6-trimethylbenzaldehyde (1) and 6-ethyl-2,4-dihydroxy-3,5-dimethylbenzaldehyde (2). Generation of DtbAΔR+TE chimeric PKSs by swapping the DtbA R domain with the AusA (austinol biosynthesis) or ANID_06448 TE domain enabled the production of two metabolites with carboxylic acids replacing the corresponding aldehydes.

Application of an efficient gene targeting system linking secondary metabolites to their biosynthetic genes in Aspergillus terreus.

Nonribosomal peptides (NRPs) are natural products biosynthesized by NRP synthetases. A kusA-, pyrG- mutant strain of Aspergillus terreus NIH 2624 was developed that greatly facilitated the gene targeting efficiency in this organism. Application of this tool allowed us to link four major types of NRP-related secondary metabolites to their responsible genes in A. terreus. In addition, an NRP affecting melanin synthesis was also identified in this species.

A versatile toolkit for high throughput functional genomics with Trichoderma reesei.

BACKGROUND: The ascomycete fungus, Trichoderma reesei (anamorph of Hypocrea jecorina), represents a biotechnological workhorse and is currently one of the most proficient cellulase producers. While strain improvement was traditionally accomplished by random mutagenesis, a detailed understanding of cellulase regulation can only be gained using recombinant technologies. RESULTS: Aiming at high efficiency and high throughput methods, we present here a construction kit for gene knock out in T. reesei. We provide a primer database for gene deletion using the pyr4, amdS and hph selection markers. For high throughput generation of gene knock outs, we constructed vectors using yeast mediated recombination and then transformed a T. reesei strain deficient in non-homologous end joining (NHEJ) by spore electroporation. This NHEJ-defect was subsequently removed by crossing of mutants with a sexually competent strain derived from the parental strain, QM9414. CONCLUSIONS: Using this strategy and the materials provided, high throughput gene deletion in T. reesei becomes feasible. Moreover, with the application of sexual development, the NHEJ-defect can be removed efficiently and without the need for additional selection markers. The same advantages apply for the construction of multiple mutants by crossing of strains with different gene deletions, which is now possible with considerably less hands-on time and minimal screening effort compared to a transformation approach. Consequently this toolkit can considerably boost research towards efficient exploitation of the resources of T. reesei for cellulase expression and hence second generation biofuel production.

Molecular genetic characterization of a cluster in A. terreus for biosynthesis of the meroterpenoid terretonin.

Meroterpenoids are natural products produced from polyketide and terpenoid precursors. A gene targeting system for A. terreus NIH2624 was developed, and a gene cluster for terretonin biosynthesis was characterized. The intermediates and shunt products were isolated from the mutant strains, and a pathway for terretonin biosynthesis is proposed. Analysis of two meroterpenoid pathways corresponding to terretonin in A. terreus and austinol in A. nidulans reveals that they are closely related evolutionarily.

Cellular localization and role of kinase activity of PMK1 in Magnaporthe grisea.

A mitogen-activated protein (MAP) kinase gene, PMK1, is known to regulate appressorium formation and infectious hyphal growth in the rice blast fungus Magnaporthe grisea. In this study, we constructed a green fluorescent protein gene-PMK1 fusion (GFP-PMK1) to examine the expression and localization of PMK1 in M. grisea during infection-related morphogenesis. The GFP-PMK1 fusion encoded a functional protein that complemented the defect of the pmk1 deletion mutant in appressorium formation and plant infection. Although a weak GFP signal was detectable in vegetative hyphae, conidia, and germ tubes, the expression of GFP-Pmk1 was increased in appressoria and developing conidia. Nuclear localization of GFP-Pmk1 proteins was observed in a certain percentage of appressoria. A kinase-inactive allele and a nonphosphorylatable allele of PMK1 were constructed by site-directed mutagenesis. Expression of these mutant PMK1 alleles did not complement the pmk1 deletion mutant. These data confirm that kinase activity and activation of PMK1 by the upstream MAP kinase kinase are required for appressorium formation and plant infection in M. grisea. When overexpressed with the RP27 promoter in the wild-type strain, both the kinase-inactive and nonphosphorylatable PMK1 fusion proteins caused abnormal germ tube branching. Overexpression of these PMK1 mutant alleles may interfere with the function of native PMK1 during appressorium formation.

Independent genetic mechanisms mediate turgor generation and penetration peg formation during plant infection in the rice blast fungus.

The first barrier to infection encountered by foliar pathogens is the host cuticle. To traverse this obstacle, many fungi produce specialized infection cells called appressoria. MST12 is essential for appressorium-mediated penetration and infectious growth by the rice pathogen Magnaporthe grisea. In this study, we have characterized in detail the penetration defects of an mst12 deletion mutant. Appressoria formed by the mst12 mutant developed normal turgor pressure and ultrastructure but failed to form penetration pegs either on cellophane membranes or on plant epidermal cells. Deletion and site-directed mutagenesis analyses indicated that both the homeodomain and zinc finger domains, but not the middle region, of MST12 are essential for appressorial penetration and plant infection. The mst12 mutant appeared to be defective in microtubule reorganization associated with penetration peg formation. In mature appressoria, the mutant lacked vertical microtubules observed in the wild type. The mst12 mutant also failed to elicit localized host defence responses, including papilla formation and autofluorescence. Our data indicate that generation of appressorium turgor pressure and formation of the penetration peg are two independent processes. MST12 may play important roles in regulating penetration peg formation and directing the physical forces exerted by the appressorium turgor in mature appressoria.